This application will define the molecular basis of heat shock protein (hsp) 27 expression in osteoblasts, based on the hypothesis that the constitutive expression of HSP 27 in osteoblasts may be a major determinant of stress tolerance as the osteoblast is relatively thermal resistant. Specific aim 1 is directed towards identifying the origins of multiple HSP 27 related proteins and transcripts expressed in stressed and unstressed osteoblasts of various differentiated states in MC3T3 cells. Genomic origins of the related transcripts will be defined by conventional screening and sequence analysis. Specific aim 2 will investigate the mechanisms underlying constitutive hsp 27 expression, heat stressed and estrogen regulated modes of hsp 27 expression. Comparisons will be made between stressed and non stressed cells. Transcriptional regulation will be examined in reporter gene constructs and comparisons will be made between MCTC929 fibroblasts, a negative control cell line that does not express hsp 27, and studies carried out in the mouse mC3T3 cell line that undergoes program differentiation. Specific aim 3 will examine potential functions of hsp 27 by determining hsp 27 interactions with other cellular proteins, based upon known functions of hsp molecules as molecular chaperones. Here unknown proteins (encoded by cDNA for nonstressed or heat stressed osteoblasts) which interact with episomal expressed hsp 27 in yeast will be identified by a karyoplasmic interaction selection strategy of cloning.